ES2363044T3 - SPECIFIC PEPTIDES FOR METALLASIS AND ITS APPLICATIONS IN DIAGNOSIS AND THERAPEUTICS. - Google Patents

Abstract

The present invention concerns peptide sequences that specifically recognize cells of human hepatic metastases. The invention comprises also the use of nucleic acids coding for such peptides, as well as conjugates and formulations of such peptides for diagnostic and therapeutic purposes.

Description

STATE OF THE INVENTION TECHNIQUE

The present invention comprises peptides that are highly specific for tumor metastatic cells, in particular liver metastatic cells, and their application in the diagnostic and therapeutic sectors.

TECHNICAL BACKGROUND OF THE INVENTION

Tumor and metastasization

Tumorigenesis is a multi-stage process, in which some cells progressively progress towards malignancy. Current knowledge in the field of neoplasia emphasizes that cancer is a disease induced by dynamic genome changes. Through these variations, tumor cells acquire independence with respect to various mechanisms that control the physiological functions of the organism. As a result, they are able to (1) grow continuously, (2) induce the recruitment of endothelial cells for the formation of new blood vessels and (3) colonize organs other than the origin.

The proliferative capacity of tumor cells is mainly due to two mechanisms. First, while normal cells need mitogenic factors to change from a still state to an active, proliferating state, in tumor cells, mutations and / or overexpression of growth factor receptors can induce the cascade of proliferation regardless of the presence of a ligand. In addition, in many cases, tumor cells acquire the ability to synthesize soluble factors, to which they are sensitive. Thus, an autocrine stimulation is established, which further increases the growth of a tumor. The other phenomenon of deregulation of the growth of cells in tumors is resistance to apoptosis (programmed cell death). This mechanism, fundamental in the growth and remodeling of organs during physiological development, is also induced in the case of non-reversible damage to the genome, to prevent the expansion of a population of aberrant cells. Some cells, however, can escape this kind of protection and become independent, thus favoring the spread of mutations and the subsequent neoplastic progression.

A primordial tumor mass is made up of a small number of cells, and could not develop above a diameter of 2 mm if it is not supported by adequate food and oxygen support. This is the phase in which an angiogenesis, that is to say the formation of new blood capillaries from previously existing blood vessels, is stimulated to a great extent by the tumor cells themselves. The imbalance between positive and negative signals of the regulation of an angiogenesis leads to the neoformation of a vascular network that penetrates and feeds the tumor mass that is actively proliferating. The blood vessels of a tumor, in addition to providing nutrition, have the function of transporting malignant cells to other districts of the body.

The progression of a tumor evolves towards a stage of irreversibility, whose characteristic peculiarity is a metastatization. In this process, pioneer cells escape from the primary tumor mass. Once they have entered the capillaries that fill the tumor, they reach the bloodstream, which transports them to regions distant from the diversion site, where they will give rise to a secondary tumor.

The development of a metastasis represents a complex biological event, related to the interactions between intrinsic factors of the organism (general conditions, integrity of the immune response) and specific characteristics of the tumor cells (location, size and histological models). From the microscopic primary site, the diffusion of tumor cells is primarily local, through a centrifugal spread. Due to the production of proteases that degrade the intercellular connections and the extracellular matrix, tumor cells invade anatomical structures and tissues that are poorly resistant (fatty tissue, nerve sheaths, bone marrow).

A first obstacle to metastatic diffusion is offered by the presence of relatively impenetrable structures, such as organ capsules, a cartilage or periosteum, the meningeal. Due to the difficulty of going beyond these limits, a metastatization in distant sites must follow stages that can be summarized as follows: (1) entry into the capillary network of a tumor by a mechanism called "intravasation", (2) transport through the bloodstream, (3) specific recognition of the destination endothelium, (4) exit from the capillary by a mechanism called “extravasation” and (5) development of a metastasis, supported by active angiogenesis.

The success of a spread depends on the anatomical characteristics and the hemodynamic factors of the host organism, and on the interactions that tumor cells can experience with the endothelium that lines the blood vessels. The most common routes of diffusion are the vessels (lymphatic and blood) and the celomatic cavities. The lymphatic vessels are penetrated quite easily, due to the absence of a basal lamina. Thus, tumor cells can easily transit into the lymph nodes, before entering the venous system through the lymphatic and venous connections. The transport within the vessels can affect both the arterial and venous system, even though the venous invasion is more common, since the venous circulation picks up the flow that leaves from certain organs. Typical examples are the systemic vein for the lung and the portal vein for the liver. Trans-celomatic dissemination concerns instead the pleural cavity of the chest and the peritoneal spaces of the abdomen and pelvis. The most commonly involved site is the peritoneum, where, after pouring fluids due to the obstruction of the hepatic veins, the tumor cells are collected in the ascites fluid. Stomach, colon, pancreas and ovarian cancers usually adopt this system.

Metastatic tumor cells express specific molecular determinants that contribute in various ways to the metastasis itself. The distribution of a metastasis is not accidental, but each tumor has preferential directions, this is known as organ-tropism. The liver is a target organ for colorectal tumors; the bones are for prostate and ovarian tumors; and the lungs are for tumors of the testicles, bones and breasts. The lungs and liver, due to their filtration function and the presence of a huge number of capillaries, can receive metastases from virtually any organ and also send tumor colonies, mainly to the brain and bones.

The liver is a common site for metastatic lesions. The reason for this must be sought and investigated in the functional and structural organization of the hepatic district. The portal vein, which evacuates blood to the abdominal viscera, represents the conduit through which cells that come from primary tumors are transported to the liver. The adhesion of circulating tumor cells to the endothelium of a liver is a critical stage for the beginning of a metastatization. Hepatic metastases develop as a consequence of the invasion of the hepatic parenchyma by these cell thrombi.

The high volume of a hepatic blood flow (approximately 25% of the cardiac flow), and the particular microscopic anatomy of the sinusoids are the factors that favor hepatic dissemination. The primary tumor can be located in the gastrointestinal tract, that is, in the colon, rectum, stomach, pancreas, biliary tract and belly. To these, you can also add tumors of the breast and lungs.

Colorectal tumor

There are different kinds of classifications that, in general, divide the progressive evolution of the disease into stages characterized by the degree of invasion of the body by that tumor. The Dukes and MAC classifications (acronym for Modified Astler-Coller = Modified Astler-Coller), proposed at the beginning of clinical studies, are now the least used. Generally, the classification of TNM (acronym for Tumor Node Metastasis = metastasis of tumor nodules) is preferred, which includes four successive stages:

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stage I: the tumor is limited to the mucosa and submucosa;

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stage II: there is extension to deeper layers of the intestinal wall;

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stage III: there is invasion of the subserosa and lymph nodes;

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stage IV: metastasis.

The most common therapeutic approaches at the present time are surgery, chemotherapy and radiotherapy. The clinical strategy class is chosen based on the stage in which the pathology is found. In general, the following protocols are used:

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stage I: a surgery (colostomy);

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stage II: surgery can be associated with chemotherapy

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stage III: surgery is associated in any case with chemotherapy;

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stage IV: a palliative treatment with surgery and / or chemotherapy.

The liver is the most frequent site of colonization by a primary colorectal cancer. Currently the only treatment that has a curative potential is the surgical removal of metastases. However, despite the increasingly effective means of liver surgery, most patients with liver metastases cannot be taken to surgery because of the extension of their tumor mass.

A different approach to cancer therapy: attacking the blood vessels of a tumor

Currently used chemotherapeutic drugs are among the drugs that have the narrowest therapeutic window in the entire medical field. As a consequence, the dose of antitumor drugs that can be administered is limited by the toxic effects on normal tissues. This difficulty can be overcome by directing the cytotoxic drugs towards the target of the tumor itself. Even though this has long been a goal in cancer biology and in cancer medicine, in reality only a few examples are now known in which the administration of a drug directed at a target is possible. For example, the use of monoclonal antibodies against tumor antigens had limited success, since only a few tumor antigens are known and the antibodies generally penetrate poorly within the tissues. In addition, since tumor cells are genetically unstable and accumulation of advantageous mutations for growth, treatments directed towards a target of tumor cells are generally followed by a clonal selection of resistant cells.

The direction towards an objective of a therapy towards the tumor vascular network allows to solve some of the problems related to a traditional therapy. Endothelial cells in the vascular system of a tumor express peculiar molecules of angiogenic vessels. The direction towards a vascular target offers several advantages. First, the endothelial lining is easily accessible. On the contrary, a drug directed towards the objective of a tumor needs to spread over long distances, penetrate within narrowly defined and fixed tumor cells and in a very dense stroma, and contrasts with a very high interstitial pressure. Secondly, since tumor cells depend on the blood supply for their growth, a therapy of a tumor directed towards the vessels does not need to lead to the destruction of all endothelial cells. Of course, a therapy directed towards an endothelial target has an intrinsic amplification mechanism. Finally, since the endothelial cells are diploid and not transformed, it is unlikely that they will loosen and release the expression of a superficial receptor or acquire drug resistance through mutations and clonal evolution. Recently, some endothelial markers have been identified. Among these molecules are some integrins, particularly αvβ3 and αvβ5 and endothelial tyrosine kinase receptors with their consanguineal ligands (VEGF receptors and the various VEGF’s, Tie1, Tie2 and angiopoietins).

Peptides that target a mouse model of human tumor: discovery of endothelial markers of tumors

Through phage display studies, performed in vivo in different animal models, peptide sequences have been identified that are capable of selectively targeting tumor vascularization. These sequences proved to be a valid instrument to characterize the endothelium of a tumor and its specific molecular determinants; and develop biotechnological applications in tumor therapy.

Thus, recurrent peptide sequences have been identified, such as RGD (Arginine-Glycine-Aspartic Acid) and NGR (Asparagine-Glycine-Arginine). The RGD motif is embedded in the sequence of several proteins of the extracellular matrix and represents its site of integration with integrins. A phage presenting the CDRGDCFC sequence, called RGD-4C, is capable of specifically targeting a breast tumor target, and selectively fixing the integrins αvβ3 and αvβ5. In vitro experiments demonstrated that peptides containing the RGD induce the adhesion of a cell with another cell, thus inducing apoptosis. Thus, it has been believed that the RGD peptide, without any further modification, can act as an antiangiogenic drug, leading to cell death after an interruption of the interactions between a cell and a matrix. Also the NGR peptide binds to integrins, although with lower affinity compared to the RGD. The specific receptor for the NGR sequence has been successively identified in another membrane protein, aminopeptidase N (APN), overexpressed in vascular structures in an active and undetectable angiogenesis in a still endothelium. It has been shown that antibodies specific to APN can inhibit a retinal neovascularization induced by hypoxia in a mouse. In the same way, mice treated with anti-APN antibodies have strongly pushed back breast tumors compared to the control set.

In another set of studies, peptides that specifically bind the NG2 proteoglycan have been identified, a homologue of HMP (acronym for human melanoma proteoglycan = proteoglycan for human melanoma) also known as Antigen Associated with a High Molecular Weight Melanoma. This proteoglycan is expressed primarily by glial progenitor cells, a muscle and a skeletal cartilage. After differentiation the expression on the surface of NG2 is lost. In adults, their presence is limited to a few vessels to active angiogenesis in some kinds of tumors, among which are a glioblastoma, a chondrosarcoma, a melanoma and some leukemias. In a mouse devoid of immunity ("naked") that carries a malignant melanoma, an anti-NG2 antibody conjugated to doxorubicin suppresses the growth of a tumor.

Peptides as antitumor drugs

Remodeling of the extracellular matrix is common both for endothelial activation and for a neoplastic invasion, and needs the action of particular enzymes called Matrix Metallo-Proteases (MMP, acronym for Matrix Metallo-Proteases). These proteases, overexpressed in the tumor, are almost absent in normal tissues, except in cases of cell migration and tissue remodeling during morphogenesis. Synthetic inhibitors of two such proteases, MMP-2 (gelatinase A; 72 Kd) and MMP-9 (gelatinase B; 92 Kd) which are the proteases most strictly involved in angiogenesis and metastatic potential, have been isolated by phage display. From this study, it has been shown that the most represented clones express the LRSGRG sequence derived from a CX6C library. Another family of proteins, identified from a CX9 collection, is the one with the HWGF motif. Soluble peptides, which contain the HWGF motif, show an inhibitory activity in vitro against MMP-9. These peptides inhibit the migration of tumor cell lineages and endothelial cells that are derived from a human umbilical cord. In vivo, they are efficient to inhibit the growth of a tumor and to prevent the appearance of metastases.

Use of peptides in biotechnologically innovative antitumor therapies

As described above, peptides that are specifically associated with tumor endothelial markers or tumor cells have been used successfully in therapeutic protocols in a mouse. A second approach has been investigated, which conjugates the RGD-4C and CNGRC peptides with the chemotherapeutic drug doxorubicin, and uses this compound for the treatment of breast tumors in mice. Animals subjected to this therapy survived for up to six months, demonstrating that this compound is capable of inhibiting both primary tumors and the development of metastases with greater efficacy and lower toxicity compared to systemically administered.

In a third set of applications, chimeric peptides have been prepared, which have two functional domains. The first one can be selectively set to the target cell and can be internalized; The latter is pro-apoptotic, not toxic in body fluids but only in the intracellular environment. There are more than 100 peptides that act causing the destruction of mitochondrial membranes and the induction of apoptosis. Among these, a sequence of 14 aa (amino acids), KLAKLAKKLAKLAK, has been selected, which proved to have a strong antibiotic potential in the form of the D-enantiomer. RGD-4C and CNGRC peptides have been coupled with this peptide. It has been found that these compounds cause mitochondrial alterations and lead to morphological variations that are typical of an apoptotic state, such as condensation and fragmentation of nuclear structures. These results have been confirmed in vivo: mice, to which the antitumor agent has been administered, show small tumors and survive for several months.

GENERAL DESCRIPTION OF THE INVENTION

The appearance of metastases is an unfavorable prognostic factor in the progression of tumors. Thus, it is essential to develop methods that allow the detection and attack of early metastases (that is, clinically undersized). In most of the cases, the histo-pathological methods currently used for a diagnosis allow to follow and monitor the location of metastases, when they are no longer suitable for treatment. In addition, from a therapeutic point of view, current approaches are limited mainly due to the non-specific toxicity of chemotherapeutic drugs.

Metastatic cells have a peculiar characteristic, compared to both the primary tumors and the tissues in which they are located. In the same way, tumor blood vessel cells (endothelial cells) are different from normal ones, which are still. In particular, significant modifications involve cell surfaces, on which molecules are expressed or modified to favor adaptation to the new environment. Classic methods of study, however, have proven ineffective in addressing the problem of the multiplicity of these modifications.

The present invention has the goal of providing a solution to overcome the deficiencies of the state of the art.

In accordance with the present invention, this goal is achieved by peptides as defined in the appended claims, particularly with peptides comprising a sequence as shown in SEQ ID NO: 1, 2, 3, 4, 6, 7, 96, 110, 111, 113, 115, 117 and 119. The invention also concerns the use of such peptides in the therapeutic and diagnostic fields. The appended claims are part of the technical advance provided here in connection with the invention.

Preferably, the present invention concerns peptides that specifically recognize liver metastatic cells. The invention also concerns the use of conjugated compounds and formulations of said peptides, when they are attached to a diagnostic agent (for example, to a brand or label) or to a therapeutic agent (for example, to a chemotherapeutic agent, a radioactive isotope or a toxin), respectively, for both in vitro and in vivo localization of liver metastatic cells and for therapy in an individual who is a tumor carrier.

The present invention can provide peptides with high binding selectivity to metastatic cells, particularly liver metastatic cells, thus allowing efficient localization of said cells both in vitro and in vivo, so that they can be used successfully both for the diagnosis as for the therapy of tumors that metastasize in the liver, more particularly primary colorectal cancers.

The peptides of the invention share the common sequence motif LRS.

Beyond the therapeutic approach, peptides that selectively recognize liver metastatic cells represent a useful means of identifying metastases themselves. The small size of these peptides is very advantageous for this class of applications. For example, conjugated peptides with radionuclides or fluorescent agents, according to the present invention, can be used in patients with hidden tumors or with non-specific radiological results. In addition, they can be used for in vivo applications, such as an MRI or CT scan, after conjugation with molecules suitable for visualization by any known visualization technique, particularly an appropriate technique for an individual body region.

Details about the formulation and administration techniques of conjugated compounds are known in the art and do not need a detailed description here, which can be dispensed with for the understanding of the invention.

The authors of the present invention used a proteomic approach (phage display) to characterize the molecular determinants expressed on the surface of cells derived from human liver metastases that are secondary to colorectal carcinomas. This technique allowed the isolation of peptides that can interact with the membrane molecules present exclusively in these cells. The identification of peptides that recognize molecular determinants that are not present in normal tissues or in the primary tumor, allows the use of said peptides for both diagnostic and therapeutic applications. From a diagnostic point of view, these peptides, labeled in an appropriate manner, can be used for the detection of liver metastases also in preclinical stages. From a therapeutic point of view, it is possible to combine them with chemotherapeutic drugs in order to adjust the protocols of an objective therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the sequences of the peptides that bind to liver metastatic cells, particularly SEQ ID NO: 1-7 represent the peptides that have been studied deeply, selected in the experiments performed with patients 16, 17 and 18; SEQ ID NO: 8-19 represent the peptides selected in the III round of selection in a sample from patient 2; SEQ ID NO: 20-39 represent the peptides selected in the selection round with a sample from patient 6; SEQ ID NO: 40-64 represent the peptides selected in the III round of selection with a sample from patient 7; SEQ ID NO: 65-78 represent the peptides selected in the II round of selection in a previous sample of patient 8; SEQ ID NO: 79-95 represent the peptides selected in the selection round in a sample from patient 16; SEQ ID NO: 96-107 represent the peptides selected in the III round of selection in a sample from patient 16; SEQ ID NO: 108-109 represent the peptides selected in the III round of selection in a sample from patient 17; SEQ ID NO: 110-118 represent the peptides selected in the III round of selection in a sample from patient 18; SEQ ID NO: 119-122 represent the peptides selected in the IV round of selection in a sample from patient 19; SEQ ID NO: 123-140 represent the peptides selected in the IV round of selection in a sample from patient 9; SEQ ID NO: 141-152 represent the peptides selected in the IV round of selection in a sample from patient 21; SEQ ID NO: 153-170 represent the peptides selected in the selection round in a sample from patient 23; SEQ ID NO: 171-186 represent the peptides selected in the IV round of selection in a sample from patient 5; and SEQ ID NO: 187-201 represent the peptides selected in the selection round in a sample from patient 8.

Figure 2 illustrates the nucleotide sequence of the primer used to sequence the oligonucleotide insert in the phage DNA;

Figure 3 illustrates an image of the poly (acrylamide) gel in which the proteins bound to the GIYRLRS peptide fused with a GST sequence have been separated.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, as a non-limiting example.

The peptides identified in this invention can be used as molecular tools in both diagnostic and therapeutic fields. It is well known that current therapeutic approaches in clinical oncology are characterized by low selectivity. A chemotherapeutic agent that circulates within the bloodstream affects, apart from the tumor masses, all populations of cells in the body that are in active proliferation. On the contrary, a peptide that is specifically recognized by specific surface receptors for a particular type of cells will be able to direct a chemotherapeutic drug preferentially towards the objective of that class of cells. The peptides described in the present invention can therefore be used successfully as a drug targeting agent towards an objective liver metastasis.

In addition, peptides labeled with a detection molecule can be used in the field of diagnosis. Currently, detection techniques are used that allow the resolution of a very early metastatic lesion from the surrounding tissues. The technology, which takes advantage of the use of labeled peptides for the detection of tumor cells, is based instead on the molecular differences that distinguish these cells from the others. Peptides according to the present invention can even detect individual human liver metastatic cells.

The data collected about the peptides of the present invention can be summarized as follows:

1) the peptides selected in the present application share a high sequence homology with each other, indicating the specificity of the selection;

2) the peptides of the present invention have a high homology with motifs present in proteins that are specific for liver tissue and / or are related to neoplastic pathologies;

3) from the fixation assays it is revealed that the peptides have a high specificity for exposed surface molecules in human liver metastatic cells, both primary and in culture, while they preferably do not show any affinity for primary liver cells normal or for cell lines of primary tumors or other kinds of metastases;

4) the peptides of the present invention are universally fixed to liver metastatic cells regardless of the metastatic stage, clinical parameters or other characteristics related to each patient, therefore being good prognostic tools - diagnostic and therapeutic candidates.

A. Definitions

As used in the present context in the specification, the articles "a" or "one" may mean "one or more". As used herein in the claims, in conjunction with the term "comprising", the words "a" or "one" may mean "one or more than one." As used herein, "other" can mean at least a second object or more than one object.

one.

 Rest of direction to a goal

A "rest of direction to an objective" is a term that encompasses various types of affinity reagents, which can be used to increase the location or fixation of a substance to a particular site in an animal, including organs, tissues, types of particular cells, diseased tissues or tumors. The targeting moieties to a target may include peptides, peptide mimetic compounds, polypeptides, antibodies, antibody-like molecules, nucleic acids, aptamers, and fragments thereof. In certain embodiments, a targeting residue to a target will increase the location of a substance in liver metastatic cells that are secondary to a colon carcinoma, through fixation to a surface protein of these cells, that is, through of binding to proteins associated with transmembrane matrices or associated with a surface or segregated or extracellular. A selective fixation of a targeting moiety to an objective of the present invention, eg a peptide or a targeting antibody to a target, as well as variants and fragments thereof, occurs when the targeting moiety to a target is set to a target (eg liver metastatic cells that is secondary to colon cancer) and not significantly fixed to unrelated cells. It is still considered that a targeting residue to a target is selectively fixed even though it is also fixed to other proteins that are not substantially homologous to the target, as long as such proteins share a homology with a fragment or domain of the target peptide of the antibody. In this case, it would be understood that an objective residue that is fixed to an objective is still selective despite presenting a certain degree of cross-reactivity. Typically, the degree of cross-reactivity can be determined and differentiated with respect to a target fixation.

2.

 Direction Peptide to a Target

A "target targeting peptide" is a peptide comprising a contiguous amino acid sequence, which is characterized by its selective location for an organ, tissue or cell type, which includes a specific fixation with an extracellular protein or molecule. , which is expressed or produced specifically in a specific tissue or in one or more type (s) of cells.

3.

 Receiver

A "receptor" for a target peptide targeting includes, but is not limited to, any molecular molecule or complex that binds a target targeting peptide. Non-limiting examples of receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, carbohydrates, multimolecular structures, and a specific conformation of one or more molecules. In preferred embodiments, a "receptor" is a molecule or complex of molecules that occur in nature, which is present on the luminous surface of cells that form blood vessels within the purpose of an organ, tissue, or cell type More specifically, a "receptor" is a molecule that occurs in nature, which is present on the luminary surface of cells that form blood vessels within the target of an organ, tissue or type of cells.

Four.

 Amino acid residue

An "amino acid residue" refers to any natural amino acid, any amino acid derivative or any amino acid mimetic compound known in the art. Protein residues are generally consecutive, without compounds other than amino acids, which interrupt the sequence of amino acid residues. In particular embodiments, the amino acid sequence may include one or more non-amino acid compounds. In particular embodiments, the amino acid sequence may include one or more non-amino acid compounds. In particular embodiments, the sequence of a peptide of the present invention may be interrupted by one or more non-amino acid compounds. Modified or unusual amino acids include, but are not limited to: Aad, 2-aminoadipic acid; EtAsn, N-ethyl-asparagine; Baad, 3-aminoadipic acid, HyI, hydroxy-lysine; BaIa, beta-alanine, beta-aminopropionic acid; AHyI, alo-hydroxy-lysine; Abu, 2-aminobutyric acid; 3Hyp, 3-hydroxyproline; 4-Abu, 4-aminobutyric acid, piperidinic acid; 4Hyp, 4-hydroxyproline; Acp, 6-aminocaproic acid, Ide, isodesmosin; Ahe, 2-aminoheptanoic acid; AIIe, alo-isoleucine; Aib, 2-aminoisobutyric acid; MeGIy, N-methyl glycine, sarcosine; Baib, 3-aminoisobutyric acid; MeIIe, N-methyl-isoleucine; Apm, 2-aminopimelic acid; MeLys, 6-N-methyl-lysine; Dbu, 2,4-diaminobutyric acid; MeVaI, N-methyl-valine; Des, desmosina; Nva, norvalina; Dpm, 2,2’-diaminopimelic acid; NIe, norleucine; Dpr, 2,3-diaminopropionic acid; Orn, ornithine; and EtGIy, N-ethyl glycine. Daminoacids are also included.

5.

 Protein or peptide

The term "protein or peptide" includes amino acid sequences consisting of at least one of the 20 common amino acids that can be found in natural proteins, or at least one modified or unusual amino acid.

6.

 Crosslinking reagents

Bifunctional "crosslinking reagents" have been widely used for a variety of purposes, including the preparation of affinity matrices, the modification and stabilization of various structures, the identification of ligands and receptor binding sites, and structural studies. Homobifunctional reagents, which carry two identical functional groups, proved to be highly efficient in inducing crosslinking between identical or different macromolecules or subunits of a macromolecule, and to link polypeptide ligands to their respective binding sites. Heterobifunctional reagents contain two different functional groups. Taking advantage of the differential reactivities of the two different functional groups, crosslinking can be controlled in both a selective and sequential manner. Bifunctional crosslinking reagents can be divided according to the specificity of their functional groups, eg, specific amino, sulfhydryl, guanidino, indole and carboxyl groups. Of these, reagents directed towards free amino groups have proven to be especially popular because of their commercial availability, their ease of synthesis and the mild reaction conditions under which they can be applied and used. A majority of heterobifunctional crosslinking reagents contain a reactive group with primary amines and a reactive group with thiols.

7.

 Antibodies

As used in the present context, the term "antibody" is intended to broadly refer to an immunological binding agent, such as IgG, IgM, IgA, IgD and IgE, or an antibody-like molecule. Generally, IgG and / or IgM are preferred, since they are the most common antibodies in the physiological situation and since they can be easily prepared in a laboratory environment. Means for preparing and characterizing antibodies are also well known in the art. "Antibody" means any molecule similar to an antibody having an antigen binding region, including antibody fragments, such as Fab ', Fab, F (ab') 2, single domain antibodies (DAB's, single domain antibodies), Fv, single chain antibodies (scFv).

8.

 Nucleic acids

"Nucleic acids" in accordance with the present invention can encode a target targeting peptide, a target targeting antibody, a therapeutic polypeptide, a fusion protein or another protein or other peptide. The nucleic acid can be derived from a genomic DNA, a complementary DNA (cDNA) or a synthetic DNA. The term "nucleic acid", as used in the present context, includes single-stranded molecules and two-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid-like compounds. It is considered that a nucleic acid within the scope of the present invention can be almost any size, determined in part by the length of the protein or peptide that has been encoded. It is considered that target targeting peptides, targeting antibodies and fusion proteins can be encoded by any nucleic acid sequence encoding the appropriate amino acid sequence. The design and production of nucleic acids that encode a desired amino acid sequence are well known to those with experience in the specialty, using standardized codon tables.

9. Supply tools

Various delivery tools can be used for administration of objective peptides according to the present invention; among others, liposomes and microemulsions systems of the types of oil in water or water in oil. Liposomes and microemulsions, and other micro-delivery systems, can be prepared by procedures well known in the art. Ligands can be fixed by covalent bonds to sites, located on the surface of liposomes. The number and surface density of these sites that can be adjusted using liposome formulations and / or specific types of liposomes. Liposome surfaces may also have sites for a non-covalent association. To form covalent conjugates of ligands and liposomes, cross-linking reagents have been studied for their effectiveness and biocompatibility. Crosslinking reagents include glutaric aldehyde (GAD), a bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE) and a water soluble carbodiimide, preferably 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) .

B. PROTEINS AND PEPTIDES

1. Peptides

The present invention involves the use of a peptide capable of selectively binding to metastatic cells, preferably liver metastatic cells.

In particular, in one embodiment, the present invention is directed to a peptide capable of selectively binding to metastatic cells, preferably to human liver metastatic cells, having the motif of LRS sequence, a length of 6 to 100 amino acids and comprising an amino acid sequence selected from the set consisting of: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119). The peptide may comprise a single copy of a sequence as defined in SEQ ID NO. 1, 2, 3, 4, 6, 7, 96, 110, 111, 113, 115, 117 and 119 or multiple identical or different copies of said sequences, optionally connected by amino acid linker sequences.

Due to their relatively small size, the objective peptides of the present invention can be synthesized in the state of solution or on solid supports, according to well known techniques. Short peptides, generally with about 6 to 35-40 amino acids, can be easily produced by these techniques. Alternatively, a recombinant cDNA technology can be used, in which a nucleotide sequence encoding a peptide of the invention is introduced into an expression vector, transformed or transfected into appropriate host cells, and cultured under conditions appropriate for the expression of proteins

The peptides of the present invention may consist of natural amino acid residues or may comprise at least one modified or unusual amino acid residue. The peptides of the present invention can be linear or cyclic peptides. The peptides of the present invention have a length of about 6 to about 100, preferably up to about 35-40 amino acids or mimetic amino acid compounds.

2.

 Mimetic Peptide Compounds

Another embodiment of the present invention involves the use of "peptide mimetic compounds". Mimetic compounds are peptides that contain molecules that mimic elements of the secondary structure of proteins. The reason that is at the base of the peptide mimetic compounds is found in the fact that the protein peptide skeleton mainly has the function of orienting the side chains of amino acids, in order to favor molecular interactions, such like those of antibodies and antigens. A mimetic peptide compound allows molecular interactions in the same way as in the natural molecule. These principles can be exploited to produce genetically engineered second generation molecules, which have most of the natural properties of the objective peptides described in the present invention, but with modified and possibly improved characteristics. An example of mimetic peptide compounds is a retroinverted peptide, formed by D-amino acids in an inverted sequence compared to the peptide sequence that it mimics. The peptide mimetic compounds of the present invention are 6 to about 100 in length, preferably up to about 35-40 amino acids or amino acid mimetic compounds.

3.

 Fusion proteins

The peptides of the present invention can also be used as one of the components of a fusion protein.

Fusion proteins comprise the entire sequence or a part of the target peptide fused together with its N and / or C end optionally through a peptide linker to a second (a) polypeptide or protein, which is heterologous. with the target peptide. For example, fusion proteins may comprise signal sequences from other proteins, to allow expression of recombinant proteins in a heterologous host. Other useful fusion proteins comprise an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. The incorporation of a dissociation site, eg a proteolytic dissociation site, next to the fusion site or in the immediate vicinity thereto, will favor the removal of the exogenous domain after purification. Other useful fusion proteins comprise functional domains, such as active enzyme sites, glycosylation domains, directional signals to cells, or transmembrane regions.

In one embodiment of the present invention, fusion proteins are produced by objective peptides fused with a protein or with a peptide with therapeutic activity. Examples of proteins or peptides, which can be incorporated into a fusion protein, include: cytostatic proteins, cytotoxic proteins, pro-apoptotic agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments of antibodies, antigens, receptor proteins, enzymes, lectins, major histocompatibility complex proteins, cell adhesion proteins and binding proteins. Such examples are not intended to be limiting, but it is understood that, according to the present invention, virtually any protein or peptide can be incorporated into a fusion protein that includes an objective peptide. The methods for producing fusion proteins are well known. Such proteins can be produced, for example, by chemical combination using bifunctional crosslinking reagents, by de novo synthesis of the entire fusion protein, or by attachment of a DNA sequence encoding the target peptide to a DNA sequence encoding the second protein or the second peptide, followed by the expression of the entire fusion protein.

4. Antibodies

In a different embodiment of the present invention, it may be desirable to produce antibodies against the target peptides that are the subject of the present invention.

For this purpose, the target peptides, or the molecules to which they are attached, can be chemically coupled, fixed, conjugated or crimped to one or more agents by means of spacer elements, polyspacer elements, or derivatized amino acids to produce a complex that It comprises at least one peptide or a target molecule that is attached to a target peptide. This can be done in a way that produces a bi- or multivalent complex, or a vaccine. The methods for producing such complexes are familiar to those skilled in the art and can be adapted for administration to a human being, that is, they can be pharmacologically acceptable. Preferred agents are vehicles, such as hemocyanin (KLH) and bovine serum albumin (BSA). The resulting antibodies can be used for both diagnosis and therapy, for example by binding and / or inhibition of functional proteins on the surface of metastatic cells.

In order to improve the efficiency of the antibody molecules, they can be fixed or converted into a complex with at least one system or one molecule, for example a molecule that allows its detection. Non-limiting examples of such molecules include enzymes, radionuclides, aptamers, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, colored particles or ligands such as biotin.

C. DIAGNOSTIC AND THERAPEUTIC CONJUGATES

In one embodiment of the present invention, it may be desirable to couple specific bioactive agents to one or more objective peptides in accordance with the present invention for specific release within an organ, tissue or cell type. Here are some examples of agents that can be coupled to target peptides according to the present invention.

Conjugates according to the present invention can be produced by direct conjugation of the target peptide to the therapeutic or diagnostic agent of interest, or by using cross-linking reagents to establish binding or binding between a peptide and the molecule of interest.

1. Cytokines

The term "cytokine" is a generic term for proteins released by a population of cells, which acts on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monochines, growth factors and traditional polypeptide hormones. Growth hormones, such as a human growth hormone, an N-methionyl human growth hormone, and a bovine growth hormone are included among the cytokines; a parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorrelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH, acronym for follicle stimulating hormone), thyroid stimulating hormone (TSH), and luteinizing hormone (LH, acronym for luteinizing hormone); liver growth factor; a prostaglandin; fibroblast growth factor; prolactin; placental lactogen; OB protein; alpha and beta tumor necrosis factors; the mullerian inhibitor substance; a peptide associated with mouse gonadotropin; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factors; transforming growth factors (TGF’s) such as TGF-alpha and TGF-beta; insulin-like growth factors l and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferons-alpha, -beta and -gamma; colony stimulating factors (CSF’s) such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (IL's) such as IL-1, IL-1alfa, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL -11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit ligand (case)

or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factors and LT. As used in the present context, the term "cytokine" includes proteins from natural sources or from a culture of recombinant cells, and biologically active equivalents of cytokines of natural sequences.

2.

 Chemokines

"Chemokines" generally act as chemoattractants in order to recruit immune effector cells to the chemokine expression site. It may be advantageous to express a particular chemokine gene in combination, with, for example, a cytokine gene, in order to intensify the recruitment of other components of the immune system to the treatment site. Chemokines include, but are not limited to, RANTES, MCAF, MIP1-alpha, MIP1-beta and IP-10. The skilled professional will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified within the term "chemokines."

3.

 Representation agents in images

In certain embodiments, the targeting molecules of an object of the present invention can be joined with imaging agents, suitable for use in imaging and diagnosing liver metastases.

Several imaging agents are well known, as are the methods for attaching them to proteins or peptides. Non-limiting examples of imaging agents include paramagnetic ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II) , neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with particular preference being Gadolinium Ions that are useful in other contexts, such as in reproduction in X-ray images, include, but are not limited to, lanthanum (III), gold (III), lead (II) and especially bismuth (III).

Radioisotopes suitable for use as imaging agents or therapeutic agents include 211statin, 14carbon, 51chromic, 36chloro, 57cobalt, 58cobalt, 67cobre, 152Eu (europium), 67galium, 3hydrogen, 123iodo, 125iodo, 131iodo, 111indium, 59 iron, 32phosphorus, 186phosphorus, 186phosphorus, 186phosphorus, 186phosphorus , 188renio, 75selenium, 35azufre, 99mtecium and 90ítrio

In certain embodiments, the proteins or peptides that are claimed can be attached to a secondary binding ligand or to an enzyme (an enzyme label or label) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, hydrogen peroxidase (horseradish) and glucose oxidase. Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such brands or labels is well known to those who have experience in the specialty.

In still other additional embodiments, a direction remainder to a target can be operatively coupled to a nanoparticle. The nanoparticles include, but are not limited to, colloidal gold and silver nanoparticles. The nanoparticles exhibit certain colors in the visible spectral region. Other additional examples of nanoparticles are magnetic nanoparticles.

4. Therapeutic agents

In certain embodiments, the therapeutic agents can be operatively coupled to a peptide or a directional fusion protein to a target for selective delivery to, for example, the tumor vasculature of liver metastases. Agents or factors suitable for use may include any chemical compound that induces apoptosis, cell death, cell stasis and / or antiangiogenesis, such as

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 Regulatory agents of programmed cell death or apoptosis. The Bcl-2 protein and other family members are involved in an apoptosis, and can be classified as agonist or antagonistic agents of apoptosis. For example, Bcl-2 and other family members (e.g., BcI-XL, Bcl-W, BcI

S, Mcl-1, A1, Bfl-1) are pro-apoptotic, while others (eg Bax, Bak, Bik, Bim, Bid, Bad, Harakiri) are anti-apoptotic.

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 Angiogenesis inhibiting agents. In certain embodiments, the present invention may concern the administration of targeting molecules to a target, which are operatively coupled with anti-angiogenic agents, such as angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibiting agents, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, thromboespondin, 2-methoxystradiol, proliferin-related protein, carboxyamidotriazole, CM101, marimastat, pentosan polysulfate, angiopoietin 2 (regenerone) , interferon-alpha, herbimycin A, PNU1451565E, a 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470 factor, Platelets 4 or minocycline.

-Cytotoxic agents. Chemotherapeutic agents (cytotoxic) can be used to treat various disease states, including cancer. Most chemotherapeutic agents fall into the categories of alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormonal agents, miscellaneous agents and any analogous compound or derivative thereof.

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 Alkylating agents Alkylating agents are drugs that interact directly with genomic DNA to prevent cells from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not specific for certain phases. An alkylating agent may include, but is not limited to, a nitrogen mustard, an ethyleneimine, a methyl melamine, an alkyl sulfonate, a nitroso-urea or a triazine. They include, but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxane), dacarbazine, ifosfamide, mechlorethamine (mustargen) and melphalan.

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 Antimetabolites Antimetabolites interrupt the synthesis of DNA and RNA. Unlike alkylating agents, they specifically influence the cell cycle during the S phase. Antimetabolites can be differentiated into various categories, such as folic acid-like compounds, pyrimidine-like compounds and purine-like compounds, and related inhibitor compounds. . Antimetabolites include, but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gencitabine and methotrexate.

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 Natural products. Natural products generally refer to compounds originally isolated from a natural source, and identified as having a pharmacological activity. Such compounds, analogous compounds and derivatives thereof can be isolated from a natural source, synthesized chemically, or recombinantly produced by any known technique for those with experience in the field. Natural products include categories such as mitotic inhibitors, antitumor antibiotic agents, enzymes and biological response modifying agents.

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 Mitotic inhibiting agents. Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit any of the protein synthesis required for cell division or mitosis. They work during a specific phase during the cell cycle. Mitotic inhibitory agents include, for example, docetaxel, ethoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine and vinorelbine. Taxoids are a class of related compounds isolated from the bark of ash, Taxus brevifolia. Taxoids include, but are not limited to, compounds such as docetaxel and paclitaxel. Paclitaxel is fixed to tubulin (at a site other than that used by vinca alkaloids) and favors the assembly of microtubules. Vinca alkaloids are a type of plant alkaloid identified as having a pharmaceutical activity. They include compounds such as vinblastine (VLB) and vincristine.

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 Antibiotics It is well known that certain antibiotics have both an antimicrobial and cytotoxic activity. These drugs also interfere with a DNA chemically inhibiting enzymes and mitosis or altering cell membranes. These agents are not specific to certain phases, so they work in all phases of the cell cycle. Examples of cytotoxic antibiotics include, but are not limited to bleomycin, dactinomycin, daunorubicin, doxorubicin (adriamycin), plicamycin (mitramycin) and idarubicin.

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 Various cytotoxic agents. Various cytotoxic agents that fall into the above categories include, but are not limited to, complex coordination compounds with platinum, anthracenediones, substituted ureas, methyl hydrazine derivatives, amsacrine, L-asparaginase, and tretinoin. Complex platinum coordination compounds include compounds such as carboplatin and cisplatin (cis-DDP). An illustrative anthracenedione is myxantrone. An illustrative substituted urea is hydroxy urea. An illustrative derivative of methyl hydrazine is procarbazine (N-methyl hydrazine, MIH). These examples are not limiting and it is considered that any cytotoxic, cytostatic or cytocidal agent can be

attached to directional peptides to a target and administered to an organ, tissue or cell type that has been established as a target within the scope of the invention.

D. NUCLEIC ACIDS

The present invention is further directed to a nucleic acid encoding a peptide capable of selectively binding to metastatic cells, which has the motive of LRS sequence, a length of 6 to 100 amino acids in sequence and comprises at least one amino acid sequence selected from the set consisting of: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6 ), GVYSLRS (SEQ ID NO. 7), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119).

Nucleic acids according to the present invention can encode a target peptide, a target antibody, a therapeutic polypeptide, a fusion protein or other proteins or peptides. The nucleic acid can be selected from a genomic DNA, a complementary DNA (cDNA) and a synthetic DNA or RNA.

In one embodiment, the present invention involves the use of vectors that express a peptide according to the present invention for gene therapy.

Vectors for gene therapy can include various transgenes, including a DNA sequence.

or RNA encoding at least one peptide or polypeptide of the present invention, operatively linked to expression control sequences.

A gene therapy can be used to express a therapeutic gene, for example to intensify or decrease neovascularization. A DNA may be in the form of a cDNA, an in vitro polymerized DNA, a plasmid DNA, parts of a plasmid DNA, a genetic material derived from a virus, a linear DNA, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, a recombinant DNA, a chromosomal DNA, an oligonucleotide, an antisense DNA, or derivatives of these sets. An RNA may be in the form of an oligonucleotide RNA, an tRNA (transfer RNA), an RNAs (small nuclear RNA), an rRNA (ribosomal RNA), an mRNA (messenger RNA), an in vitro polymerized RNA, a Recombinant RNA, chimeric sequences, an antisense RNA, an siRNA (a small interfering RNA), ribozymes, or derivatives of these sets. An antisense polynucleotide is a polynucleotide that interferes with the function of a DNA and / or RNA. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA, RNA and the like. An siRNA comprises a double-stranded structure, typically containing 15-50 base pairs and preferably 21-25 base pairs and having a nucleotide sequence that is identical or almost identical to that of a target gene or RNA expressed within the cell. An interference can result in a suppression of the expression. The polynucleotide can also be a sequence whose presence or expression in a cell alters the expression or function of cellular genes or RNA. In addition, DNA and RNA can be single-stranded, double-stranded, triple-stranded or quad-stranded.

MATERIALS AND METHODS

The phage display methodology

Phage display is a technique developed in the mid-1980s by George Smith of the University of Missouri. The principle is to select some peptides from a collection, or from a library, in which virtually all possible permutations of amino acids are represented. Such peptides are selected based on their ability to specifically set an objective of any nature and complexity considered. The phage display methodology involves rounds of screening and amplification of fixed particles, with the goal of obtaining a reduction in diversity and an increase in specificity for fixation.

The construction of a phage library involves the use of M13 filamentous bacteriophages that can infect Escherichia coli bacteria. A peculiar feature of these phages is that they have a single-stranded circular DNA genome, which can be manipulated with molecular biology techniques. In such a library, the peptides are derived from the transcription and translation of random exogenous oligonucleotides, which are cloned into the viral DNA located upstream from the gene for the capsid protein pIII. Bacteria are transformed with these artificial constructions by electroporation; thus, they will produce a population of recombinant phages, each of which will include a different peptide in the form of a fusion with the pIII protein. With this system, it is possible to produce a library with a diversity of approximately 106-108 peptides, in which each sequence is represented up to 100-1,000 times. If the degeneration of the sequence is complete (Xn, in which X is = any amino acid and n is the number of amino acids), each of the 20 amino acids has the same theoretical probability of being included within the sequence. Another possibility is to establish fixed positions for an amino acid. Libraries are frequently characterized by cysteines located in preferred positions, next to both ends of the peptide or interspersed within random residues, between which intermolecular disulfide bridges are formed that make the peptide circulate. The circularization of the insert allows a better exposure of the sequence.

Abbreviations and solutions

BCE

3-amino-9-ethyl carbazole

Amp

ampicillin

BSA

bovine serum albumin

DMEM

Dulbecco's minimum essential medium

DMEM / FCS / HEPES DMEM mixture with a high glucose content / 2% FCS / 20 mM HEPES DMSO dimethylsulfoxide

DTT dithiothreitol DTA ethylene diamine tetraacetic acid FCS fetal calf serum GST glutathione sulfo-transferase HEPES N-2-hydroxy ethyl piperazino-N’-2-ethyl sulfonic acid HRP Horseradish peroxidase Kan kanamycin

IPTG isopropyl-β-thio galactoside LB broth from Luria Bertani PAF paraformaldehyde

PBS phosphate buffered saline solution, 150 mM NaCl,

10 mM KH2PO4, pH 7.40. PEG / NaCl 20% poly ethylene glycol – 8,000, 4 M NaCl PMSF methyl sulfonyl fluoride SDS 40 mM TAE sodium dodecyl sulfate Tris-HCI, 0.12% acetic acid, 1 mM EDTA Buffer 50 mM of Tris-HCI, pH 7.40, 150 mM NaCl, 5% glycerol,

2 mM DTT Buffer H 10 M Tris-HCI, pH 7.40, 10 mM NaCl, 10 mM PMSF TBS Tris buffered saline, 150 mM NaCl, 2.8 mM KCl, 25 Mm Tris pH base

7.40 TBS-T TBS-0.1%, Tween-20 TB Terrific Broth Tetracycline Tet

10 mM TE from Tris-HCI, 1 mM EDTA Reagents Disposable plastic material: Falcon, Eppendorf. Media and other reagents for cell culture: DMEM with high glucose content and RPMI-1640: from Sigma;

Ham DMEM and F12: from Biowhittaker Europe; FCS: from Gibco; collagenase: from Roche; L-glutamine and penicillin / streptomycin solution: from Biowhittaker Europe; broths and antibiotics for bacteria cultures: LB: from Sigma; TB: from Gibco; Kan and Tet: from Sigma; immunohistochemical reagents: from DAKO Cytomation.

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Surgical specimens

Surgical samples are derived from patients undergoing surgical operations of the Institute for Cancer Research and Treatment (IRCC Institute for Cancer Research and Treatment), Candiolo (TO), Italy, Division of Oncological Surgery. Written consent for participation in this study was obtained from all donors.

A sample of a normal liver and a liver metastasis was obtained for each patient. The samples were morphologically different in size, appearance, color, vascularization, the presence of necrotic regions, and the accumulation of lipid conglomerates (which is an index of the degree of degeneration induced by steatosis). The differences in the tissues are related to the different phase of disease progression, to the site of metastasis of the primary tumor, to other possible causes or diseases that would have occurred in the pathogenic process, or to other reasons related to individual variability.

The samples were prepared immediately after a surgical extraction, in order to disintegrate the tissues and extract the individual cells, in which the experiments were to be performed. All manipulations were performed under a laminar flow under sterile conditions. The samples were cut and chopped with a scalpel in a small volume of a PBS. The suspension, collected in a PBS, was centrifuged for 3 minutes at 100 rpm (revolutions per minute) at room temperature and the sediment was resuspended in 5 ml of the collagenase (0.25% by weight / volume in DMEM ). Digestion of tissue fragments was performed by incubating this suspension for 2 hours at 37 ° C while stirring. The sample was centrifuged again, in order to remove all the particles with a size below that of the cells (lipid conglomerates, cell portions) or also smaller cells, of hematopoietic origin. The sediment was washed twice in a PBS. The cells were filtered on filters having a diameter of 45 µm, counted in a Bürker chamber and resuspended at a concentration of 106 / ml in a mixture of DMEM / FCS / HEPES.

On microscopic examination, after a breakdown of the tissues and a purification of the cells, the primary cell population appeared as heterogeneous and other types of cells other than hepatocytes and tumor cells could be distinguished. Among these, there are red blood cells (red blood cells) and other cells of hematopoietic origin; fibroblasts derived from parenchymal connective structures; endothelial cells that line the blood vessels of the analyzed tissue.

Cell lineages

For the experiments of the present invention the human cell lineages indicated in the Table were used

one.

Table 1 Media for cell culture

Cell lineage

Description

SW480

Primary Colorectal Cancer (ATCC CCL228)

SW620

Metastasis of lymph nodes from a colorectal cancer (ATCC CCL227)

NCI-H630

Hepatic metastasis from a colorectal cancer (ATCC CRL5833)

HepG2

Primary liver cancer (ATCC HB8065)

AGS

Primary Stomach Cancer (ATCC CRL1739)

NCI-H87

Hepatic metastasis from stomach cancer (ATCC CRL5822)

Capan 2

Primary Pancreas Cancer (ATCC HTB80)

Capan 1

Liver metastasis from pancreatic cancer (ATCC HTB79)

BT-474

Primary Breast Cancer (ATCC HTB20)

MCF-7

Pleural effusion from breast cancer (ATCC HTB22)

A549

Primary Lung Cancer (ATCC CCL185)

NCI-H1688

Liver metastasis from lung cancer (ATCC CCL257)

For the maintenance and growth of cell lineages different culture media were used, depending on the type of cells:

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SW480, SW620, HepG2, BT-474 and MCF-7 cells were cultured in a DMEM, with 10% FCS, 20 mM HEPES, L-glutamine (40 mM), penicillin (200 U / ml) and streptomycin ( 200 µg / ml).

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NIC-H630, NCI-H87, NCI-H1688, Capan-1 and Capan-2 cells were

    cultured in RPMI-1640, with 10% FCS, L-glutamine (40 mM),

penicillin (200 U / ml), and streptomycin (200 µg / ml).

-A549 and AGS cells were cultured in Ham F12, with 10% FCS,

L-glutamine (40 mM), penicillin (200 U / ml) and streptomycin (200 µg / ml).

Cell cultures

Cultures were started from cells stored in liquid nitrogen in an FCS solution with 10% DMSO. The cells, after rapid thawing at 37 ° C, were grown in 100x20 mm dishes, in a humidified incubator at 37 ° C with 5% CO2. The complete replacement of the culture medium was done every 3-4 days. When a confluence of 80-90% was reached, the cells were washed in a PBS and separated by incubation with a solution of 0.05% trypsin and 2 mM EDTA at 37 ° C for 3 minutes. An excess volume of the medium with 10% FCS was then added and the cells were collected by precipitation at 1,000 rpm for 3 minutes. The supernatant material was removed, the sediment was resuspended and distributed in aliquots in 4 new plates.

For phage display experiments, the cells were washed in a PBS with 10 mM EDTA, and incubated in the same solution for 3 minutes at 37 ° C. Then the cell suspension was harvested in a PBS in a total volume of 10 ml. After a count in the Bürker chamber, the cells were resuspended in a mixture of DMEM / FCS / HEPES with a final concentration of 1x106 / ml.

Phage Libraries

For phage display experiments, two cyclic libraries of types CX7C and CX3CX3CX3C and a linear library of type CX9 were used. In these libraries the insert is expressed in 5 identical copies as a fusion peptide next to the N-terminus of the pIII protein. The cysteine of the insert, close to the surface of the capsid, is covalently fixed to the phage protein and, in the case of the CX7C library, can form a disulfide bond with the cysteine on the opposite side. As a consequence of this, the peptide is cyclized. In the CX3CX3CX3C library, instead, different combinations of disulfide bridges can be formed, leading to multiple cyclisations and the exposure of tripeptide motifs.

The CX9 library is linear and there is no cyclization, except in a case where the last amino acid is a cysteine, as well. Phage libraries are stored at 4 ° C in TBS, at a concentration of 1010-1012 UT / ml.

Broths and plates for bacteria cultures

LB: this medium was supplemented either with Kan, to a final concentration of 20 µg / ml, for the amplification of the K91kan strain of the Escherichia coli bacteria, or with both Kan and Tet, both at a rate of 20 µg / ml , for the amplification of bacteria after infection.

TB: This broth was used to make the K91kan bacteria competent for an infection, and was supplemented with Kan, to a final concentration of 20 µg / ml.

Plates: the bacteria were amplified in Petri dishes on a semi-solid substrate, which was composed as follows: LB with 15% by weight / volume of a bacteriological agar, and Kan (20 µg / ml) for the growth of K91kan, or Kan (20 µg / ml) and Tet (40 µg / ml) for the growth of infected bacteria.

Selection of libraries in cells

For all processes regarding phage display, the protocols known in the literature were used. In particular, the protocol for total cell separation by adsorption is derived from some methods described, but was adapted to the system under analysis, after several tests, with the goal of optimizing the application.

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First round. One microliter of the library was incubated with 5x105 recently collected metastatic cells, in a total volume of 500 µl in a mixture of DMEM / FCS / HEPES, for 16 hours at 4 ° C, with gentle agitation. Four washes were then performed in the same medium in order to eliminate weakly fixed phages or phages that have been left in solution. The washings were performed in 1 ml of the same medium.

Successive rounds In the successive rounds of selection, 50 µl of the phages obtained from round I were incubated with 5x105 cells of a normal liver from the same patient in 500 µl of a mixture of DMEM / FCS / HEPES. This negative preselection operation lasted 1 hour at room temperature with gentle agitation, and was repeated twice. The supernatant material was then divided into two parts, which were added to 5x105 cells, taken from cells either from the normal liver or from a liver metastasis, respectively. The two cell suspensions were incubated for 2 hours at 4 ° C with gentle agitation. A few washes followed, as described. The fixed phages were collected infecting competent bacteria.

Bacteria infection and phage amplification

The bacteria were grown in 10 ml of a TB with Kan, at 37 ° C while stirring for 2-3 hours, until they reached the optical density of 1.5-2.0 at the wavelength of 600 nm. Then, 1 milliliter of competent bacteria was added to 100 µl of the cell suspension, after washing. The infection lasted 1 hour at room temperature. At the end of the incubation, a part of the bacteria were spread, in duplicate, on Petri dishes with an LB agar and with Tet, and incubated for 16 hours at 37 ° C. This system allows only bacteria infected with phages to grow, since only phages carry resistance to this antibiotic. The UT (titration or titration units) related to the fixed substrate phage were evaluated by counting the colonies of each plate. Here, we refer to this value with the term "exit."

The remaining part of the bacteria was added to 10 ml of a LB with Tet and Kan and grown for 16 hours at 37 ° C while stirring.

Phage Purification

This process was used to purify both phage populations that were derived from the selection rounds and from the clones of a single phage. The bacteria culture was centrifuged at 5,000 rpm for 10 minutes at 4 ° C to remove bacteria. The phages, which were now in the supernatant material, were precipitated with 0.15 volumes of a PEG / NaCl mixture for 1 hour at 4 ° C and collected by centrifugation at 6,000 rpm for 15 minutes at 4 ° C. After having decanted the supernatant material, the sediment was compacted by further centrifuging at 6,000 rpm for 5 minutes at 4 ° C and then resuspended, by stirring for 10 minutes, in 500 µl of TBS. To eliminate waste, the suspension was then centrifuged at 12,000 rpm for 10 minutes at room temperature. The phage population was collected and stored at 4 ° C.

Phage titration

The degree allows you to evaluate the amount of starting UT for each round or the title of the individual clones (whose amount we refer to as “entry”). To make the assessment from the original phage suspension, the dilutions described in Table 2 have been made.

Table 2

Sample 1)

1 µl of phage suspension + 99 µl of PBS (1x10-2 dilution)

Sample (2)

10 µl of the sample (1) + 90 µl of PBS (1x10-3 dilution)

Sample (3)

10 µl of the sample (2) + 90 µl of PBS (1x10-4 dilution)

Sample (4)

10 µl of the sample (3) + 90 µl of PBS (1x10-5 dilution)

Sample (5)

10 µl of the sample (4) + 90 µl of PBS (1x10-6 dilution)

Sample (6)

20 l of the sample (5) in a new tube

Sample (7)

2 µl of the sample (6) + 18 µl of PBS (1x10-7 dilution)

Sample (8)

2 µl of the sample (7) + 18 µl of PBS (1x10-8 dilution)

100 µl of the samples (6), (7) and (8), that is, those of dilutions 1x10-6, 1x10-7 and 1x10-8 were spread on Petri dishes with agar and Tet. The plates were incubated for 16 hours at 37 ° C. The total number of UT was then evaluated by counting the colonies, and referred to the total volume.

Single clone fixation assays

These tests were performed with an input of 109 TU of each clone, in cells from the liver metastasis cell lineage (target) and in cells from a normal liver (negative control). The output of these experiments was normalized by fixing a phage without an insert, fd-tet, giving a measure of the non-specific interaction due to the phage itself. The increase in fixation was evaluated as the ratio between the standardized output of the target and the normalized output of the negative control. All experiments were repeated at least three times, when possible in terms of material availability.

Isolation and amplification of the clones

When a significant increase was observed between the number of phages fixed to metastatic cells compared to those fixed to cells of a normal liver, individual clones were isolated to identify the sequence of their insert and to assess its specificity of fixation. For amplification of the clones, bacteria from individual colonies were grown in 5 ml of LB with Kan and Tet for 16 hours at 37 ° C while stirring. Then the phages were purified as described.

To mechanically disintegrate the phage capsid, resin beads, called Strataclean beads by the manufacturer, were used. Before use, the beads were resuspended in TBS in a 1: 1 volume / volume ratio. For each clone, 200 µl of a phage suspension was added to 10 µl of beads and vorted for 30 seconds. After centrifugation at 400 rpm for 3 minutes, 195 µl of a supernatant material was collected and subjected to the same disintegration cycle. Finally, 150 µl of the supernatant material was filled and completed to 410 µl with TE, and the DNA was precipitated by incubation with 0.1 volumes of sodium acetate pH 5.5 and 2.2 volumes of 100% ethanol. The DNA was collected by centrifugation for 10 minutes at 12,000 rpm, washed in 70% ethanol and resuspended in ultrapure H2O. The amount of DNA was evaluated both by reading its absorbency at a wavelength of 260 nm and by electrophoresis in 1% agarose gels in APR.

Sample preparation for sequencing

Ten microliters of the solution, corresponding to approximately 800 ng of the phage DNA, were incubated with 3 pmol of the following primer:

5'-CCCTCATAGTTAGCGTAACG-3 '(SEQ ID NO. 202), which corresponds to an area located immediately downstream from the oligonucleotide insert.

Sequence Analysis

To translate the nucleotide sequences into peptide sequences, we have used the DNAsis V2.5 software program.

Immunohistochemical protocol with phages

Tissue samples were embedded in OCT and stored at –80 ° C. For the experiment, they were cut using a cryostat at –20ºC.

The tissues were cut into 10 µm slices. These slices were then treated with a PBS for 5 minutes until the OCT had been completely removed. The tissues were fixed in 4% PAF in PBS for 10 minutes at room temperature and then washed for 5 minutes in a PBS and incubated with 50 mM NH4Cl in a PBS for 20 minutes.

Tissue peroxidases were then deactivated by treating with 3% H2O2 in H2O for 10 minutes in the dark at room temperature. A wash followed for 5 minutes in a PBS.

The nonspecific interaction sites were blocked by incubating the samples in the "DAKO block" reagent for 30 minutes at room temperature. Phages (from 1x106 to 5x106 total UT) diluted in the "DAKO diluent" reagent were then added; incubation lasted overnight at 4 ° C. After 4 washes for 5 minutes each in TBS, the samples were stained with a rabbit polyclonal M13 phage antibody (Sigma B7786), diluted at 1: 500 in the "DAKO diluent" reagent for 1 hour at room temperature .

The labeling was performed using a secondary "EnVision DAKO" anti-rabbit antibody, developed with the AEC substrate for 5 minutes and followed by a contraction with Mayer's hematoxylin.

Purification of the peptide fused with GST

Some of the selected peptides were produced in Escherichia coli as a fusion protein with GST, using classical purification protocols.

Preparation and lysis of bacteria:

1. bacteria are inoculated and allowed to grow overnight in a 20 TB / Amp broth at 30 ° C;

2. the bacteria are transferred to 300 ml of a TB / Amp broth at 30 ° C; it shakes 5 for 1 hour;

3.

 IPTG (to a final concentration of 1 mM) is added and incubated for 2 hours;

Four.

 centrifuge at 5,000 rpm for 15 minutes at 4 ° C;

5.

 the bacteria are resuspended in 10 ml of buffer A;

10 6. centrifuge at 3,000 rpm for 20 minutes at 4 ° C;

7.

 the sediment is suspended again in 5 ml, the bacteria are treated with four pulses, each for 20 seconds with 35% of the power;

8.

 centrifuge at 11,000 rpm for 20 minutes at 4 ° C and the material is collected

15 supernatant Preparation of a glutathione-agarose resin

9.

 250 µl of the resin is hydrated in distilled H2O, rotating for 1 hour;

10.

 the resin is washed 3 times in buffer A and finally returned to

  suspend in an equal volume of buffer A. 20 Purification of recombinant proteins:

eleven.

 250 µl of the resin is added to the sample of step 8;

12.

 it is rotated at 4 ° C for 1 hour;

13.

 wash 3 times in buffer A;

14.

 the concentration is evaluated by electrophoresis followed by

25 staining with Coomassie blue. Polymerization mixture for SDS-polyacrylamide gel electrophoresis (Table 3). Table 3 Staining with Coomassie Blue

12% gel in operation

acrylamide / bis-acrylamide (4 ml) 1.5 M Tris pH 8.8 (3.75 ml) 10% SDS (0.1 ml) double-distilled water (2.15 ml) ammonium persulfate (100 mg / ml) (33 µl) TEMED (8 µl)

5% stacking gel

Acrylamide / bis-acrylamide (0.8 ml) 0.5 M Tris pH 6.8 (650 ml) 10% SDS (0.05 ml) double distilled water (3.55 ml) ammonium persulfate (100 mg / ml) (30 µl) TEMED (8 µl)

one.

 The gel is incubated with Coomassie blue for 30-45 minutes in soft

 agitation,

2.

 fade into a mixture of 45% methanol and 10% acetic acid;

5 3. It is rehydrated in water, eventually dried on paper.

Cell lysis

Twenty 100x20 mm plates of HepG2 (of hepatoma) or NCI-H630 (secondary liver metastases for

a colorectal carcinoma) were mechanically separated in a PBS and resuspended in 2 volumes of the

buffer H, with 10% glycerol and 0.1% Nonidet-P40. These suspensions were incubated for 30 minutes at 4 ° C

10 with stirring and then centrifuged at 2,500 rpm for 30 minutes at 4 ° C. The protein concentration was evaluated by means of the BCA case (from Pierce), following the manufacturer's instructions.

Demolition test

GST peptides bound to the resin were incubated overnight at 4 ° C with milk and washed 7 times in buffer A. 10 mg of the total protein lysate was incubated with 12 µg of the GST-resin complex at 4 ° C

15 for 1 hour twice to remove proteins that are fixed in a non-specific manner to GSTs or resin. The demolition assay was performed on the unbound proteins, with 12 µg of the GST-peptidine complex, overnight at 4 ° C.

After 4 washes in buffer A, the proteins bound to the GST-peptide-resin complex were eluted in 20 mM glutathione for 30 minutes at 4 ° C and collected by centrifugation at 3,000 rpm for 2 minutes at 20 4 ° C.

The supernatant material was charged on a 10% polyacrylamide denaturing gel. Then this gel is

stained with a Coomassie blue solution and the specific bands were collected and analyzed by

mass spectrometry and micro-sequencing (with standardized standard protocols).

RESULTS

25 Search for peptide motifs that are specific to human liver metastases

In order to find peptides that specifically bind human liver metastases, scrutiny was performed in phage libraries about suspended cells that are derived from a normal liver and from metastatic samples, which have been surgically removed from the liver of the patients. In this phase, 11 pairs of samples from different patients were used (patients 2, 5, 6, 7, 8, 16, 17, 18, 19, 21, 23). In

30 almost all samples, liver metastases were secondary to primary tumors of the colon or rectum, excluding patient 8, who had a cerebral hemangioma as a primary tumor (Table 4).

Table 4

Patient

Sex Age Place1 TN2 Bookmark3 Virus4  Necrosis5

2

F 60 Colon T3N0 CEA Do not 40%

5

M 46 Colon T4N2 CEA Do not 60%

6

M 72 Colon T3N2 CEA Do not fifty%

8

F 31 Brain Do not

7

M 64 Colon T3N1 GICA Do not fifteen%

16

 M Four. Five Colon T4N2 Do not twenty%

17

 M 70 Colon T3N0 Do not 80%

18

 F 62 Colon T4N0 CEA, GICA Do not 60%

19

 M 66 Colon T3N0 CEA Do not fifty%

twenty-one

 M 59 Straight T3N0 Do not <5%

2. 3

 F 49 Colon Do not fifty%

1: primary tumor site;

2: classification according to TN of the primary tumor;

3: tumor markers;

4: evidence of hepatitis B or C virus in the liver;

5: percentage of necrosis within metastases.

Table 4 shows the clinical parameters of the patients used for selection. For patients 2, 6, 7 (2 experiments), 16, 17, 18, 19 and 21 the CX7C library was used; for patient 8, both CX7C and CX3CX3CX3C libraries were used in samples of two different metastases; for patient 23 the CX9 library was used. For each experiment, 4 rounds of selection and amplification were performed.

Analysis of the peptide sequences obtained in the screening experiments

In each experiment in which we observed a significant increase in the binding ratio to liver metastatic cells and to the negative control (macroscopically healthy liver tissue), 20 clones of phage were amplified and purified. The DNA of each clone was purified and sequenced to deduce the peptide motif. The selected peptides are shown in Figure 1.

Some sequences are particularly represented, both in the same experiment and in experiments performed on samples from different patients. In the experiments performed on patients 2, 5, 6, 7, 8, 21 and 23, peptides were shared that shared common sequences, particularly tri / tetra-peptide motifs (among which are GGG, RGL, GRL, GSG , LGR, GLS, SAD, YEG and GSGS). In the experiments performed on patients 16, 17 and 18 we have found more repeated sequences. In these experiments there were also reasons that had a high homology with those described above. The most repeated peptide is the LRS.

Analysis of the selected sequences

The focus was on the study of the sequences obtained in experiments 16, 17 and 18, in particular: ARPGLRS (SEQ ID NO. 1); MRYALRS (SEQ ID NO. 2); LRPGLRS (SEQ ID NO. 3); LRSGSGS (SEQ ID NO. 4); VRSGRGS (SEQ ID NO. 5); GIYRLRS (SEQ ID NO. 6); and GVYSLRS (SEQ ID NO. 7). In order to identify homologies between sequences between these peptides and these proteins, a search was made in the BLAST database. From these analyzes it appeared that a significant number of peptides share sequence homologies with extracellular matrix proteins and with cell adhesion / mobility molecules.

Fixation experiments in cell lineages

To assess whether the selected inserts were specific ligands for a superficial determinant peculiarly expressed in liver metastases, the 7 clones were tested in the cell lineages described in Table 1. A summary of the results is shown in Tables 5 and 6.

In this study model, selected sequences of peptides are not fixed to cells that are derived from primary tumors (with the exception of BT-474). On the contrary, these sequences are preferably fixed to cells derived from liver metastases (6 of 7 clones are fixed to liver metastatic cells that are secondary to a primary colon tumor), 3 of 7 clones are fixed to liver metastatic cells that They are secondary to a stomach or lung tumor.

Table 5.

SEQ ID NO.

Hep-G2 Liver tumor SW480 Colon tumor SW620 Lymph node metastasis of colon tumor NCI-H630 Hepatic metastasis of colon tumor AGS Stomach Tumor NCI-N87 Stomach Tumor Liver Metastasis

one

- - - - - -

2

- - + + - +

3

- - - + - -

4

- - - + - +

5

- - - + - +

6

- - - + - -

7

- + - - - -

Table 6.

SEQ ID NO.

Capan-2 Pancreas tumor Capan-1 Hepatic metastasis from a pancreas tumor BT-474 Breast tumor MCF-7 Pleural effusion of breast tumor A549 Lung tumor NCI-H1688 Hepatic metastasis of lung tumor

one

- - - - - +

2

- - - - - -

3

- - - - - -

4

- - + + + +

5

- - + - - -

6

- - - + - +

7

- - - - + -

Fixation experiments in primary cells

In order to assess whether the selected inserts were specific for ubiquitous surface determinants in human liver metastases, the 7 selected clones were tested in primary liver metastatic cells, compared to a normal liver of the same patients. For these fixation assays, samples from 9 patients (20, 21, 22, 25, 26, 27, 28, 31, 32) were used, under the same conditions as described for cell lineages. A summary of the results is shown in Table 7.

Table 7.

SEQ ID NO.

P nº20 P nº21 Pcs. nº22 P nº25 P nº26 P nº27 P nº28 P nº31 P nº32

one

 + + + + + +

2

 + + - - + +

3

+ + + + + + +

4

 - + + + + + +

5

 - + + + + +

6

+ + + + + +

7

+ + + + + + +

In the first experiments, due to the low numbers of cells, related to the establishment phase of the purification process, the fixation of only some clones was evaluated. In general, 3 tests have been performed for each sample. From all these experiments it appears that the less functional clone

5 as a universal diagnostic marker is the one that presents the MRYALRS sequence (SEQ ID NO. 2), which gave negative results in two trials (27, 28), while the clones that worked in all the samples are the ones that expose the sequences ARPGLRS (SEQ ID NO. 1), LRPGLRS (SEQ ID NO. 3), GIYRLRS (SEQ ID NO. 6) and GVYSLRS (SEQ ID NO. 7). It is interesting to note that, in recent cell experiments, increases in fixation are much higher than in those performed with cultured cell lineages.

10 Experiments of fixation overlap in tissue samples

Fixation overlapping tests with phages having the sequences GIYRLRS (SEQ ID NO. 6) and GVYSLRS (SEQ ID NO. 7) were performed on 64 tissue samples (tumor and metastasis tissues from different 37 different patients): 18 samples of liver metastases and healthy blood tissue; 4 samples of a primary colon tumor; 2 samples of a primary rectal tumor; 2 samples of

15 a healthy colon; 3 samples of a primary breast tumor; 6 samples of a primary ovarian tumor with inbred omental metastases (one with sigma metastases): 2 secondary lung metastases for colorectal tumors and 2 secondary lung metastases for a renal tumor. The results of all tests are shown in Table 8.

Table 8

Type of fabric

Outcome % of positive samples

secondary liver metastasis for a colorectal tumor

+++ 75

healthy colon

- 0

healthy liver

- 0

primary colorectal tumor

- 0

primary ovarian tumor

- + 10

omental metastasis of ovarian tumor

- + 10

metastatic ovarian tumor sigma

- 0

lung metastasis of colorectal tumor

- 0

lung tumor metastasis

- 0

20 Purification of receptors The search for molecules specifically present on the surfaces of metastatic cells was performed by demolition experiments, using those of NCI-H630 (as a substrate, which is 23

positive for the fixation of 6 of 7 phages) and HepG2 (as a control, which is negative for the fixation of all phages) The demolition was performed using the GIYRLRS peptide (SEQ ID NO. 6), present in the form of a fusion with the GST protein. This experiment was repeated three times. A denaturing polyacrylamide gel is shown in Figure 3, in which the GIYRLRS-GST-bound proteins have been separated and stained with Coomassie blue. In the figure: MM, molecular weight markers, HepG2, lysate material of HepG2 cells; NCIH630, lysate material of NCI-H630 cells; the numbers 250, 150, 100, 75, 50, 37, 25 indicate the standard molecular weights; the numbers from 1 to 9 indicate the bands analyzed. Proteins were identified by mass spectrometry.

The subject of the following paragraphs is also covered by the present invention:

one.

A peptide capable of selectively binding to metastatic cells, having the motif of the LRS sequence, a length of 6 to 100 amino acids and comprising an amino acid sequence selected from the set consisting of: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7), LRSGRGS (SEQ ID NO 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119).

2.

A peptide of paragraph 1, wherein said metastatic cells are human liver metastatic cells.

3.

A peptide of any one of paragraphs 1 or 2, which is a cyclic peptide.

Four.

A peptide of any one of paragraphs 1-3, comprising at least one modified amino acid, an unusual amino acid and / or an amino acid in conformation D.

5.

A conjugate comprising at least one peptide capable of selectively binding to metastatic cells of any one of paragraphs 1-4, and at least one molecule.

6.

A conjugate of paragraph 5, wherein said at least one molecule is selected from a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptotic agent, an anti-angiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytostatic agent, a peptide, a protein, an antibody and an antibody fragment such as Fab fragment.

7.

A conjugate of paragraph 6, wherein said anti-angiogenic agent is selected from the set consisting of thromboespondin, angiostatin, pigmented epithelial derived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibiting agents , tissue metalloproteinase inhibitors, interferons, interleukin 12 (IL-12), platelet factor 4, IP-10, 2-methoxystradiol, a protein related to proliferin, carboxyamidotriazole, CM101, marimastat, pentosan polysulfate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, a 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, a proteasome inhibiting agent, a kinase inhibitor agent, a peptide signaling, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline.

8.

A conjugate of paragraph 6, in said pro-apoptotic agent is selected from the set consisting of etoposide, ceramide sphingomyelin, Bax, Bid, Bik; Bad, caspasa-3, caspasa-8, caspasa-9, fas, ligand of fas, fadd, fap-1, tradd, faf, rip, reaper, apoptin, the enzyme converting interleukin-2 and annexin V.

9.

A conjugate of paragraph 6, wherein said cytokine is selected from the set consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18 , gamma interferon (IF-gamma), IF-alpha, IF-beta, tumor tumor necrosis factor (TNF-alpha), and GM-CSF granulocyte and macrophage colony stimulating factor.

10.

A conjugate of paragraph 5, wherein said at least one molecule is selected from a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic pearl, a nanoparticle, a yeast cell, and a mammalian cell .

eleven.

A conjugate of paragraph 10, wherein said virus is selected from an adenovirus, a retrovirus, a virus associated with adeno and a lentivirus.

12.

A conjugate of paragraph 5, wherein said at least one molecule is a diagnostic agent.

13.

A conjugate of paragraph 12, wherein said diagnostic agent is a diagnostic agent for in vivo use.

14.

A conjugate of paragraph 13, wherein said diagnostic agent is selected from paramagnetic ions or radioisotopes.

fifteen.

A conjugate of paragraph 12, wherein said diagnostic agent is a diagnostic agent for in vitro assays.

16.

A nucleic acid encoding a peptide capable of selectively binding to metastatic cells, having the motif of LRS sequence, a length of 6 to 100 amino acids in sequence and comprising at least one amino acid sequence that is selected from the set that is It comprises: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS ( SEQ ID NO. 7), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115 ), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119).

17.

A formulation comprising at least one peptide capable of specifically binding to metastatic cells, of any one of paragraphs 1-4.

18.

A formulation of paragraph 17, wherein said at least one peptide is conjugated to a drug.

19.

A formulation of paragraph 18, wherein said drug is a therapeutic agent capable of having a cytotoxic, cytostatic, pro-apoptotic or anti-angiogenic effect on liver metastatic cells.

twenty.

A formulation of paragraph 18, in said drug is an alkylating agent, an anti-metabolite or an antibiotic.

twenty-one.

A formulation of paragraph 18, wherein said at least one peptide is conjugated to a diagnostic agent.

22

A formulation of paragraph 21, wherein said diagnostic agent is a diagnostic agent for in vivo use.

2. 3.

A formulation of paragraph 21, wherein said diagnostic agent is a diagnostic agent for in vitro assays.

24.

A formulation of any of paragraphs 17-23, which is a pharmaceutical formulation.

25.

A formulation of any of paragraphs 17-24, which includes at least one acceptable vehicle and / or excipient.

26.

Use of a peptide of any of paragraphs 1-4 for the production of a diagnostic formulation intended for the location of metastatic cells in an individual with a tumor, particularly with a colon tumor.

27.

Use of paragraph 26, wherein said metastatic cells are liver metastatic cells.

28.

Use of paragraph 26 or 27, in which said formulation is intended for in vivo use.

29.

Use of paragraph 26 or 27, in which said formulation is intended for in vitro tests.

30

Use of a peptide of any of paragraphs 1-4 for the production of a medicament intended for antitumor therapy in an individual carrying a tumor.

31.

Use of a nucleic acid of paragraph 16 for the production of a medicament intended for antitumor therapy in an individual carrying a tumor.

32

Use of paragraph 31, wherein said antitumor therapy is a gene therapy.

33.

An in vitro method for obtaining a peptide capable of selectively binding to a metastatic cell, which has an LRS sequence motif and is 6 to 100 amino acids in length, the method (1) comprising contacting the metastatic cell or a tissue containing metastatic cells with a plurality of phages, in which each phage has heterologous peptide sequences incorporated into a capsid protein, (2) removing phages that are fixed to cells or tissues, (3) isolating phages that are they attach to the cell or tissue, and optionally (4) identify heterologous peptide sequences.

3. 4.

An in vitro procedure of paragraph 33, wherein said metastatic cells are hepatic metastatic cells.

35

An in vitro procedure of paragraph 34, wherein said liver metastatic cells are derived from a primary colorectal tumor.

36.

An in vitro method of any one of paragraphs 33-35, wherein said peptide comprises at least one sequence selected from the set consisting of: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2 ), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS

(SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119).

BIBLIOGRAPHY

one.

 Arap, W., Pasqualini, R. & Rouslahti, E. Chemotherapy targeted to tumor vasculature [Chemotherapy directed at a tumor vasculature]. Curr. Opin. Oncol. 10 560-565 (1998).

2.

 Pasqualini, R., Arap, W. Rajotte, D. & Rouslahti, E. On Phage display: a laboratory manual (presentation coordinators Barbas, CF, Editors Barbas, CF, Burton, DR, Scott, JK & Silverman, GJ) 124 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000).

3.

 Del Gatto, A. et al. Novel and selective alpha (v) beta3 receptor peptide antagonist: design, synthesis, and biological behavior [New and selective antagonist agent of the selective alpha (v) beta3 receptor peptide: design, synthesis, and biological behavior ]. J Med Chem 49, 3416-20 (2006).

Four.

 Colombo, G. et al. Structure-activity relationships of linear and cyclic peptides containing the NGR tumor-homing motif [Relationships between structure and activity of linear and cyclic peptides containing the messenger motif of NGR tumors]. J. Biol Chem 277, 47891-7 (2002)

5.

 Corti, A. & Ponzoni, M. Vascular tumor targeting with tumor necrosis factor alpha and chemotherapeutic drugs [Direction towards a vascular target of tumor with necrosis factor of alpha tumors and chemotherapeutic drugs]. Ann N and Acad Sci 1028, 104-12 (2004).

6.

 Curnis, F. et al. Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vesels, epithelia, and myeloid Cells [Differential fixation of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia and myeloid cells] . Cancer Res 62, 867-74 (2002)

7.

 Di Matteo, P. et al. Immunogenic and structural properties of the Asn-Gly-Arg (NGR) tumor neovasculature-homing motif [Immunogenic and structural properties of the Asn-Gly-Arg (NGR) messenger to the neovasculature of a tumor]. Mol Immunol 43, 1509-18 (2006).

8.

 Koivunen, E., Wang, B. & Rouslahti, E., Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library [Isolation of a highly specific ligand for alpha 5 beta 1 integrin from a phage display library]. J. Cell Biol 124, 373-80 (1994).

9.

 Pasqualini, R. et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis [Aminopeptidase N is a receptor for messenger peptides to tumors and an objective to inhibit angiogenesis]. Cancer Res 60, 722-7 (2000).

10.

 Pastorino, F. et al. Vascular damage and anti-angiogenic effects of vessel-targeted tumor liposomal chemotherapy [Effects of vascular and anti-angiogenic damage of chemotherapy with liposomes directed towards a target of vessels of a tumor], Cancer Res 63, 7400 -9 (2003).

eleven.

 Burg, MA, Pasqualini, R., Arap, W. Ruoslahti, E. & Stallcup, WB NG2 proteoglycan-binding peptides target tumor neovasculature [Some peptides that fix NG2 proteoglycans target the target of a neovasculature of a tumor] Cancer Res. 59, 2869-2874 (1999).

12.

 Koivunen, E. et al. Tumor targeting with a selective gelatinase inhibitor [Direction to the target of a tumor with a selective gelatinase inhibitor]. Nat. Biotechnol. 17, 768-774 (1999).

13.

 Ellerby, H. M. et al. Anti-cancer activity of targeted pro-apoptotic peptides [Anticancer activity of pro-apoptotic peptides targeting a target]. Nat. Med. 5, 1032-1038 (1999).

14.

 Scott, J. K. & Smith, G.P. Searching for peptide ligands with an epitope library [Search for peptide ligands with an epitope library]. Science 249, 386-390 (1990).

fifteen.

 Smith G.P. & Scott, J. K. Libraries of peptides and proteins displayed on filamentous phage [Peptide and protein libraries presented in a filamentous phage]. Methods Enzymol. 217, 228-257 (1993).

Claims (14)

one.

A peptide capable of selectively binding to metastatic cells, preferably human liver metastatic cells, having the motif of the LRS sequence, a length of 6 to 100 amino acids and comprising an amino acid sequence selected from the set consisting of: ARPGLRS ( SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7) ), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119).

2. A peptide of claim 1, which is a cyclic peptide.

3.

A peptide of any one of claims 1 or 2, comprising at least one modified amino acid, an unusual amino acid and / or an amino acid in conformation D.

Four.

A conjugate comprising at least one peptide capable of selectively binding to metastatic cells of any one of claims 1-3, and at least one molecule.

5.

A conjugate of claim 4, wherein said at least one molecule is selected from a drug, a chemotherapeutic agent, a radioisotope, an anti-apoptotic agent, an antiangiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytostatic agent, a peptide, a protein, an antibody, an antibody fragment such as a Fab fragment, a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic pearl, a nanoparticle, a yeast cell and a mammalian cell, in which

said anti-angiogenic agent is preferably selected from the set consisting of thromboespondin, angiostatin, the pigmented epithelial factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen inhibitor agents, tissue metalloproteinase inhibitors, interferons, interleukin 12 (IL-12), platelet factor 4, IP-10, 2-methoxystradiol, proliferin-related protein, carboxyamidotriazole, CM 101, marimastat, pentosan polysulfate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E , a 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, a proteasome inhibitor agent, a kinase inhibitor agent, a signaling peptide, accutin, cidofovir, vincristine , bleomycin, AGM-1470, platelet factor 4 and minocycline, said pro-apoptotic agent is preferably selected from the group consisting of etoposide, ceramide sphingomyelin, Bax, Bid, Bik; Bad, caspasa-3, caspasa-8, caspasa-9, fas, ligand of fas, fadd, fap-1, tradd, faf, rip, reaper, apoptin, interleukin-2 converting enzyme and annexin V, said cytokine is preferably selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL10, IL-11, IL-12, IL-18, interferon gamma (IF-gamma), IF -alpha, IF-beta, tumor necrosis factor alpha (TNFalfa), and GM-CSF (the granulocyte and macrophage colony stimulating factor) and said virus is preferably selected from an adenovirus, a retrovirus, a virus associated with adeno and a lentivirus.

6.

A conjugate of claim 4, wherein said at least one molecule is a diagnostic agent intended for in vivo use, preferably selected from paramagnetic ions or radioisotopes, or for in vitro assays.

7.

A nucleic acid encoding a peptide capable of selectively binding to metastatic cells, which has the motif of the LRS sequence, a length of 6 to 100 amino acids in sequence and comprises at least one amino acid sequence selected from the set consisting of: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. .7), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119).

8.

A formulation comprising at least one peptide capable of specifically binding to metastatic cells of any one of claims 1-3.

9.

A formulation of claim 8, wherein said at least one peptide is conjugated with a drug, preferably with a therapeutic agent capable of having a cytotoxic, cytostatic, proapoptotic or anti-angiogenic effect on liver metastatic cells, an alkylating agent , an anti-metabolite or an antibiotic.

10.

A formulation of claim 8, wherein said at least one peptide is conjugated to a diagnostic agent intended for use in vivo or for in vitro assays.

eleven.

A formulation of any one of claims 8-10, which is a pharmaceutical formulation that preferably includes at least one vehicle and / or an acceptable excipient.

12.

Use of a peptide of any one of claims 1-3, for the production of a diagnostic formulation intended for in vivo use or for in vitro assays for cell localization

5 metastatic, preferably liver metastatic cells, in an individual with a tumor, particularly with a colon tumor. 13. Use of a peptide of any one of claims 1-3 or of a nucleic acid of claim 7, for the production of a medicament intended for antitumor therapy in an individual carrying a tumor. 14. An in vitro procedure for obtaining a peptide capable of selectively binding to metastatic cells, preferably hepatic metastatic cells, which are particularly derived from a primary colorectal tumor, which has an LRS sequence motif and is 6 to 100 in length. amino acids, in which the method comprises (1) contacting the metastatic cell or a tissue containing metastatic cells with a plurality of phages, in which each phage has heterologous peptide sequences 15 incorporated into a capsid protein, (2) remove phages that are not fixed to cells or tissues, (3) isolate phages that are fixed to the cell or tissue, and optionally (4) identify the sequences of heterologous peptides 15. An in vitro method of claim 14, wherein said peptide comprises at least one sequence selected from the set consisting of: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID 20 NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7), LRSGRGS (SEQ ID NO. 96), RREGLRS ( SEQ ID NO. 110), SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115), VRPGLRS (SEQ ID NO. 117) and LRSGRGS (SEQ ID NO. 119 ).